Deamidated interferon-beta

ABSTRACT

Interferon-β protein analogs in which the asparagine at position 25, numbered in accordance with native interferon-β, is deamidated exhibit a biological activity of native human interferon-β at an increased level and do not require HA for protein stabilization. The deamidated product is suitable for large scale manufacturing for incorporation in HA-containing or HA-free therapeutics for treatment of diseases including multiple sclerosis. An endoproteinase-C peptide map technique that produces a fingerprint profile for proteins using an enzymatic digest followed by RP-HPLC is also useful in quality control as an ID and/or quantitative test for the deamidated products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/626,837, filed Nov. 10, 2004, titled DEAMIDATED INTERFERON-BETA,the disclosure of which is incorporated herein by reference in itsentirety and for all purposes.

BACKGROUND

1. Technical Field

This invention is in the general area of biologically active proteinchemistry. More specifically it relates to mutationally and chemicallyaltered Interferon-β analogs that differ from the native protein bysubstitutions, deletions or modifications of cysteine, asparagine andother residues.

2. Background Art

Interferon-β has been found to be useful in the treatment of humandisease, in particular multiple sclerosis. Multiple sclerosis (MS) is achronic, often disabling disease of the central nervous system thatoccurs when a protective sheath surrounding nerve fibers breaks down.About thirty percent of MS patients suffer from a relapsing-remittingform of the disease in which symptoms disappear totally or partiallyafter a flare-up and are followed by a period of stability that can lastfor months or years. Administration of beta interferon (Interferon-β orIFN-β) has been demonstrated to reduce the frequency of MS flare-ups. Asa result, Interferon-β based pharmaceuticals have become a valuable toolin management and treatment of MS.

Recombinant DNA (rDNA) techniques have been developed to facilitate thelarge scale manufacturing of Interferon-β based pharmaceuticals. Oneproblem in particular that needed to be addressed by these techniqueswas that human beta interferon, the amino acid sequence of which isprovided in FIG. 1 (SEQ ID NO: 1), contains cysteine residues atpositions 17, 31, and 141, Gene (1980) 10:11-15 and Nature (1980)285:542-547), at least some of which are nonessential to their activitybut are free to form undesirable intermolecular or intramolecular links.In the course of the microbial preparation of IFN-β by rDNA techniques,it has been observed that dimers and oligomers of IFN-β are formed inextracts containing high concentrations of IFN-β due to thisintermolecular linking. This multimer formation renders purification andisolation of IFN-β very laborious and time-consuming and necessitatesseveral additional steps in purification and isolation procedures suchas reducing the protein during purification and reoxidizing it torestore it to its original conformation, thereby increasing thepossibility of incorrect disulfide bond formation. In addition, thismultimer formation has been associated with low specific biologicalactivity.

In order to address these issues, refined rDNA techniques have beendeveloped to alter microbially produced biologically active IFN-βprotein analogs in a manner that does not affect their activityadversely, but reduces or eliminates their ability to formintermolecular crosslinks or intramolecular bonds that cause the proteinto adopt an undesirable tertiary structure (e.g., a conformation thatreduces the activity of the protein). Directed mutagenesis techniqueshave been successfully used to form mutationally altered biologicallyactive protein analogs (a “protein analog” refers herein to a syntheticprotein in which one or more amino acids has been genetically and/orchemically modified and that retains a biological activity of the parentprotein) that retain a desired activity of their parent proteins butlack the ability to form intermolecular links or undesirableintramolecular disulfide bonds. Synthetic protein analogs of IFN-βbiologically active protein which have the cysteine residue at position17 deleted or replaced by another amino acid have been found to have thedesired activity and characteristics.

In particular, Interferon-β 1b (IFN-β 1b), a synthetic, recombinantprotein analog of IFN-β, is a biologically active protein which has thecysteine residue at position 17 replaced by a serine residue has beenmade. As a microbially produced protein, IFN-β 1b is unglycosylated. Italso has an N-terminal methionine deletion. IFN-β 1b has been formulatedinto a successful pharmaceutical marketed as Betaseron® that has beenshown to be effective for treatment and management of MS. This proteinanalog, materials and techniques for its manufacture, its formulation asa therapeutic and its use to treat MS are described and claimed in anumber of U.S. patents and applications including application Ser. No.435,154, filed Oct. 19, 1982; U.S. Pat. No. 4,588,585, issued May 13,1986; U.S. Pat. No. 4,737,462, issued Apr. 12, 1988; and U.S. Pat. No.4,959,314, issued Sep. 25, 1990; each of which is incorporated byreference herein for their disclosure of these features.

Large scale manufacturing of IFN-β for pharmaceuticals is also conductedfrom mammalian sources, in particular Chinese hamster ovary (CHO) cells.This IFN-β analog, referred to as IFN-β 1a, lacks the Ser17 mutation ofIFN-β 1b and is glycolsylated. IFN-β 1a is formulated into therapeuticproducts marketed as Avonex® and Rebith®.

As with most therapeutics, there is a continual desire to identify andmanufacture more potent biologically active agents. It the case of IFN-βbased pharmaceuticals, a IFN-β analog with increased biological activitywould be desirable.

In addition, some IFN-β pharmaceutical formulations, includingBetaseron®, contain human albumin (HA or HSA), a common proteinstabilizer. HA is a human blood product and is in increasingly lowsupply. Accordingly, more recently there has been a desire for HA-freedrug formulations, and a stable and effective HA-free IFN-β formulationwould be desirable.

SUMMARY OF THE INVENTION

The present invention addresses these needs by providing deamidatedInterferon-β. The deamidated IFN-β is a human interferon-β proteinanalog in which the asparagine at position 25, numbered in accordancewith native interferon-β, is deamidated. The deamidated product exhibitsa biological activity of native human interferon-β at an increased leveland does not require HA for protein stabilization.

In a specific embodiment, the deamidated IFN-β is a synthetic humaninterferon-β 1b protein analog in which cysteine at position 17,numbered in accordance with native interferon-β, is deleted or replacedby a neutral amino acid, in particular serine, and the asparagine atposition 25, is deamidated such that it becomes a cyclic imide,aspartate or iso-aspartate residue. The deamidated product exhibits thedesired biological activity of native human interferon-β (e.g., cellularcytopathic effects or antiproliferative activity, such as have beenshown to be correlated with reducing the frequency of multiple sclerosisflare-ups) at an increased level relative to its IFN-β parent protein.In addition, the enhanced biological activity is observed in an HA-freeformulation of the protein analog.

Formulations of the active protein analog compounds in therapeuticcompositions and methods of making and use are also provided.

In addition, an endoproteinase-C peptide map technique that produces afingerprint profile for proteins using an enzymatic digest of a reducedprotein sample at relatively low pH, followed by chromatographicresolution of peptide fragments, useful in quality control as an ID testfor the deamidated products is provided.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the amino acid sequence of IFN-β.

FIG. 2 is diagram of the amino acid sequence of IFN-β 1b indicating thesite and nature of the deamidation in accordance with the presentinvention.

FIG. 3 is diagram illustrating the Asn deamidation pathway withreference to INF-β Asn25 deamidation in accordance with the presentinvention.

FIGS. 4-11 show plots of the potency versus the amount of deamidatedIFN-β 1b in various HA-free IFN-β stability drug substance and productlots in accordance with one aspect of the invention.

FIG. 12A shows Glu-C peptide maps of HA-Free IFN-β stability samples inaccordance with one aspect of the invention.

FIG. 12B shows an expanded view a portion of the map of FIG. 12A.

FIG. 13 shows the RP-HPLC profile of an HA-Free IFN-β control sample(time (min) vs. response (mV)).

FIG. 14 shows the RP-HPLC profile of the stability sample of the HA-freeIFN-β 1b drug product lot in accordance with one aspect of theinvention.

FIG. 15 shows a plot of the CPE Activity for RP-HPLC fractions of anHA-Free IFN-β stability sample in accordance with one aspect of theinvention.

FIG. 16A shows a plot of results of an antiproliferative activityagainst Hs294T for RP-HPLC fractions of an HA-Free IFN-β stabilitysample in accordance with one aspect of the invention.

FIG. 16B shows a plot of results of an antiproliferative activityagainst Daudi for RP-HPLC fractions of an HA-Free IFN-β stability samplein accordance with one aspect of the invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The compounds, compositions, materials and associated techniques anduses of the present invention will now be described with reference toseveral embodiments. Important properties and characteristics of thedescribed embodiments are illustrated in the structures in the text.While the invention will be described in conjunction with theseembodiments, it should be understood that the invention it is notintended to be limited to these embodiments. On the contrary, it isintended to cover alternatives, modifications, and equivalents as may beincluded within the spirit and scope of the invention as defined by theappended claims. In the following description, numerous specific detailsare set forth in order to provide a thorough understanding of thepresent invention. The present invention may be practiced without someor all of these specific details. In other instances, well known processoperations have not been described in detail in order not tounnecessarily obscure the present invention.

INTRODUCTION

The present invention provides deamidated Interferon-β. The deamidatedIFN-β is a human interferon-β protein analog in which the asparagine atposition 25, numbered in accordance with native interferon-β, isdeamidated. Asn deamidates to Asp or iso-Asp via a cyclic imideintermediate. The deamidated product exhibits a biological activity ofnative human interferon-β at an increased level and does not require HAfor protein stabilization. Formulations of the active protein analogcompounds in therapeutic compositions and methods of making and use arealso provided.

A “protein analog” refers herein to a synthetic protein in which one ormore amino acids has been genetically and/or chemically modified andthat retains a biological activity of the parent protein, such ascellular cytopathic effects or antiproliferative activity. Suchbiological activity has been shown to be correlated with other specificbiological activities, such as reducing the frequency of multiplesclerosis flare-ups.

In a specific embodiment, the deamidated IFN-β is a purified andisolated synthetic human interferon-β 1b protein analog in whichcysteine at position 17, numbered in accordance with native interferon-βis deleted or replaced by a neutral amino acid, in particular serine,and the asparagine at position 25, is deamidated. In specificembodiments, the Asn25 is deamidated to an aspartate, iso-aspartate orcyclic imide residue (e.g., IFN-β_(ser17,asp25,) IFN-β_(ser17,iso-asp25)or IFN-β_(ser17,cyclic-imide25), respectively). The deamidated productexhibits a biological activity of native human interferon-β at anincreased level relative to IFN-β 1b. In addition, the enhancedbiological activity is observed in an HA-free formulation of the proteinanalog, enabling an HA-free IFN-β 1b therapeutic.

In the synthetic protein analog of the invention, the cysteine 17residue may be replaced by a serine, threonine, glycine, alanine,valine, leucine, isoleucine, histidine, tyrosine, phenylalanine,tryptophan or methionine. In a specific embodiment, the substitution isserine 17. The asparagine 25 residue has been replaced by an aspartate,iso-aspartate or a cyclic imide. Referring to FIG. 2, the primary (aminoacid sequence (SEQ ID NO: 2)) and secondary (folding, cross-linking)structure of a IFN-β 1b protein analog in accordance with the inventionis illustrated. At position 25, the native Asn residue is deamidated.The deamidation of the asparagine occurs via a cyclic imideintermediate. This pathway is illustrated in FIG. 3. As described infurther detail below, Glu-C peptide map results indicate that the majorforms of deamidation are iso-Asp (IFN-β_(ser17,iso-asp25)) and cyclicimide (IFN-β_(ser17,cyclic-imide25)).

The protein analog may be made by a combination of recombinant syntheticand chemical modification techniques. Initially, the IFN-β 1b syntheticprotein analog is typically made by recombinant DNA directed mutagenesistechniques, as described in the patents referenced above in theBackground section of the application. Directed mutagenesis techniquesare well known and have been reviewed by Lather, R. F. and Lecoq, J. P.in Genetic Engineering Academic Press (1983) pp 31-50.Oligonucleotide-directed mutagenesis is specifically reviewed by Smith,M. and Gillam, S. in Genetic Engineering: Principles and Methods, PlenumPress (1981) 3:1-32.

The IFN-β 1b protein analog is then subjected to a chemical treatmentthat deamidates the asparagine residue at position 25. There are anumber of possible deamidation techniques that may be effective andsuitable for adoption in large scale pharmaceutical manufacturing andany technique that accomplishes the deamidation while retaining a nativebiological activity, preferably an increased biological activity, may beused. Broadly speaking, deamidation of IFN-β proteins can be achieved byincubation at moderate to high temperature (e.g., about 25-60° C.) and avariety of pHs, from low (e.g., about 0-4) moderate (e.g., about 4-10)to high (e.g., about 10-14) with reaction times from about 1 minute toabout 90 days or more, depending upon conditions. For example,deamidation may be accomplished by incubation of IFN-β (e.g., IFN-β 1aor IFN-β 1b) for up to 60° C.; or between about 25 and 40° C., forexample about 40° C., at about pH 4 for at least 24 hours, for exampleup to 40 days. The reaction time may be decreased and/or the reactiontemperature decreased by raising the pH, for example to a neutral tobasic pH of about 7 up to 14, for example for about 8 to 12, e.g., about8.5. In one example, the biological activity (CPE) of a sample of INF-β1b increases almost 2-fold after treatment at pH 8.4 at 2-8° C. for 14days to about 4.5 IU/mg. Substantial activity increases have also beenobserved for treatments at moderate (e.g., room temperature) to high(e.g., 37-40° C.) temperature for 14-40 days.

The techniques for preparing the deamidated IFN-β protein analog mayproduce a product that is partially or substantially pure. For example,at least 25%, at least 50%, at least 75% or substantially all of thesynthetic protein analog in the product may be deamidated at position25, numbered in accordance with native interferon-β. Where less thansubstantially all of the product is deamidated, the product maynevertheless show increased biological activity and HA-free stability.In some instances, it may be desirable to purify and isolate thedeamidated protein analog(s) in the product. This purification andisolation may be achieved by cationic exchange HPLC for example usingthe following conditions:

Chromatography Stationary Phase: Pharmacia Mono S HR 5/5, or equivalent;

Eluent Buffer: 20 mM Tris-HCl, pH 7.0 with 0.5% Empigen;

Gradient: NaCl linear gradient up to 200 mM or higher in the eluentbuffer.

For manufacturing, this technique can be conducted on a large scale.

In a preferred embodiment, when the synthetic protein analog ismicrobially produced, it is unglycosylated. Also, the protein analog hasan N-terminal methionine deletion. In other embodiments, the proteinanalog may be produced in mammalian cells and thus be glycosylated.

As described in further detail below, various activity assaysdemonstrate that deamidated IFN-β 1b protein analogs have increasedbioactivity relative to their parent IFN-βproteins. And the stabilityresults have been obtained with HA-free samples indicating that thedeamidated IFN-β protein analogs of the present invention are suitablefor HA-free formulation as a therapeutic. To form a therapeuticcomposition, the protein analog, partially or substantially pure asdescribed above, can be admixed with a pharmaceutically acceptablecarrier medium, such as are well known for this type of therapeuticproduct.

While it is an advantageous property of compositions of the presentinvention that they exhibit HA-free stability, the deamidation may alsobe conducted for HA-containing formulations and these are not excludedfrom the scope of the present invention. In addition, while theinvention is primarily described here with reference to IFN-β 1b proteinanalogs, the invention is also applicable to other IFN-β analogs,including IFN-β 1a analogs.

The IFN-β protein analogs and compositions of the present inventionexhibit biological activity that suggest utility in therapeutics in anumber of applications including regulating cell growth in a patient,treating a patient for viral disease and stimulating natural killer cellactivity in a patient. One particular use is for treating multiplesclerosis in a patient, in particular relapsing-remitting MS. In thisrespect, therapeutics in accordance with the present invention areuseful in treatment for reducing the frequency of multiple sclerosisflare-ups. The increased level of biological activity of the deamidatedIFN-β protein analogs indicates enhanced effects as therapeutics, forexample in the treatment and management of MS.

According to another aspect of the present invention, anendoproteinase-C (Glu-C) peptide map technique is provided. The peptidemap produces a fingerprint profile for proteins using an enzymaticdigest of a reduced protein sample at a relatively low pH, followed byliquid chromatographic (e.g., RP-HPLC) resolution of the digestfragments. The peptide mapping may be used in quality control as an IDtest for deamidated IFN-β protein analog product in accordance with theinvention. It is also a powerful tool to monitor minor primary structuremodifications in a protein from events such as clipping, mutation anddegradation due to oxidation or deamidation.

One variant of deamidated INF-β is known to contain cyclic imide, anintermediate form of deamidation. This cyclic imide from is found inincreased amounts in stability samples. Most enzymes including Lys-C,which is used in a conventional peptide map for IFN-β, optimally digestproteins at neutral to high pH. At neutral to high pH, the cyclic imideis unstable and further deamidation is artificially induced. Therefore,maintaining the sample at a low pH environment during reduction anddigestion is necessary to monitor the native level of both cyclic imideand other deamidation froms (Asp, iso-Asp) in the sample.

The endoproteinase-C (Glu-C) peptide map is coupled with a reducingagent that is functional at a pH of below 8, e.g., in the range of about3-8, for example about 3, 4, 5, 6, 7 or 8 or pHs intermediate those. Asuitable reducing agent is tris-(2-carboxyethyl) phosphine (TCEP). Otherreducing agents functional at a pH of about 3-8 may be used, such asdithiothreitol (DTT), 2-mercaptoethanol, cysteine, reduced glutathione,2-mercaptoethylamine and thioglycollic acid. The Glu-C peptide map hastwo optimal pHs, pH 7.8 and pH 4.0, for its enzymatic activity. As notedabove, TCEP is known to be functional at pH below 8.0. In oneembodiment, the new Glu-C peptide map uses the sample preparationincluding both reduction by TCEP and digestion at low pH, 4.0, which isat the optimal pH range to preserve the native level of deamidatedforms, e.g., cyclic imide, in the sample. This new map technique may beused to characterize the digest fragment containing deamidated forms,including cyclic imide, in IFN-β samples.

IFN-β samples may be tested by the Glu-C peptide map to identify thedeamidation site and its form (e.g., Asp, Iso-Asp, cyclic imide).Protein samples in buffered formulations (e.g., 0.5 ml protein sample ata concentration of 0.1 to 10, e.g., 0.5 mg/ml in formulation buffer of 2to 500 mM buffer, or in some cases 50 to 100 mM, of a salt such asaspartate, bicarbonate (e.g., ammonium bicarbonate), carbonate (e.g.,ammonium carbonate), acetate (e.g., ammonium acetate), phosphate (e.g.,sodium phosphate), citrate, formate, succinate, MES, PIPES, ACES, MOPS,MOPSO, HEPES, TES, TRIS-HCl, BIS-TRIS, BIS-TRIS Propane, ADA, BES,DIPSO, TAPSO, HEPPSO, POPSO, EPPS, TEA, etc. the appropriate selectionand use of which will be known to those of skill in the art for thedesired pH (e.g., 2 mM aspartic acid, pH 4)) can be reduced using TCEP,for example using a 1:2 to 1:30, e.g., 1:3, 1:4, 1:5, 1:10 or 1:20 molarratio of protein to TCEP, such as 1:10. The sample is incubated, forexample at about 30-40° C. (e.g., 37° C.), until the sample material isreduced. Suitable incubation times may be from about 5 minutes to 24hours depending on sample lability, for example 3 hours. The reducedmaterial is subsequently digested with Glu-C, for example 1 to 10, e.g.,4 mg/ml, at a 1:1 to 20:1 (or any suitable intermediate ratio including2:1, 3:1, 4:1, 10:1, etc.), e.g., 5:1, mass ratio of protein to Glu-Cand incubated, for example at about 30-40° C. (e.g., 37° C.), until thesample material is digested. Suitable digestion times may be from about5 minutes to 24 hours depending on sample lability, for example 4 hours.The peptide fragments can be resolved by liquid, e.g., RP-HPLC,chromatography.

A specific peptide map experiment and its results are described in theExamples section below.

EXAMPLES

The following examples illustrate aspects of the present invention, butare not intended in any way to limit the invention. In various of theseexamples, a distinction is made between the cyclic imide and otherdeamidated forms (Asp and iso-Asp), the two main distinguishable speciesthat showed increase of potency. All are deamidated forms, but where adistinction is drawn between the cyclic imide and the other deamidatedforms (Asp and iso-Asp) the former is sometimes referred to as “cyclicimide” while the latter is sometimes referred to as “deamidated” or“deamidation,” particularly in the figure labels.

Example 1 Potency Increase in HA-Free IFN-β 1b Stability Sample

Summary

A potency increase has been observed in HA-Free IFN-β 1b stabilitysamples. The cytopathic effects (CPE) bioassay showed a potency increasein HA-Free IFN-β 25° C. stability samples in the course of time (T=0-6months). The final deamidation product and its intermediate, cyclicimide, have also been observed to be increased in 25° C. stabilitysamples.

Glu-C peptide mapping identified the deamidation site at Asn25 andrevealed that the major forms of deamidation in the HA-free IFN-β 1bstability samples were the iso-Asp and cyclic imide analogs, while theAsp form is slightly increased.

The results obtained from RP-HPLC method indicated that the deamidatedforms (cyclic imide, Asp and iso-Asp) significantly increased in HA-freeIFN-β stability sample.

The deamidated forms in the HA-free IFN-β stability sample showed higherbiological activity than the parent (amidated) IFN-β by CPE andantiproliferative assays.

Based on these findings, the deamidation (forming Asp, iso-Asp andcyclic imide analogs) is considered to enhance a biological activity ofHA-free IFN-β. Therefore, a deamidated form of IFN-β can be prepared inorder to enhance a biological activity of IFN-β-based therapeutics,either HA-free or HA-containing. The deamidated analogs can be preparedby incubating an IFN-β solution (either HA-free or HA-containing) atmoderate to high temperature, and/or at low, moderate or high pH. Thedeamidated products of this invention will reduce the required clinicaldose and increase the stability of liquid IFN-β formulations at roomtemperature. By lowering the clinical dose, the proportion of patientsexperiencing an adverse immune reaction (e.g., neutralizing antibodies),is reduced.

Data substantiating the invention obtained from various stabilitystudies and analyses of IFN-β 1b preparations are provided below:

1. HA-Free IFN-β Stability Data

As shown below in Table 1 (Drug Substance: in 2 mM Aspartic acid, pH4.0) and Table 2 (Drug Product: in 2 mM Aspartic acid, pH 4.0, 9%Trehalose), the cytopathic effects (CPE) bioassay showed a potencyincrease in HA-free IFN-β 1b 25° C. stability samples in the course oftime (T=0-6 months):

TABLE 1 CPE Bioassay Stability Data for the HA-Free Interferon beta 1bDrug Substance Analytical Method: CPE Bioassay Acceptance Criteria:1.3-5.1 × 10⁷ IU/mg Lot No.: Test Run TA2040 TA2085 Test Run TA2040TA2085 Description: 6 ml; 2 mg/ml 6 ml; 2 mg/ml 6 ml; 2 mg/ml 6 ml; 2mg/ml 6 ml; 2 mg/ml 6 ml; 2 mg/ml Study No.: 1402 1411 1414 1403 14121415 Storage: 5° C. 5° C. 5° C. 25° C./60% 25° C./60% 25° C./60% RH RHRH Orientation: Upright Upright Upright Upright Upright Upright IU/mg ×IU/mg × IU/mg × Months 10⁷ 10⁷ 10⁷ IU/mg × 10⁷ IU/mg × 10⁷ IU/mg × 10⁷0   2.8 2.8 2.9 2.8 2.8 2.9 1.5 ND ND ND 4.2 3.5 3.6 3   3.0 2.9 2.8 4.64.2 4.1 4.5 ND ND ND 5.7 4.9 5.0 6   3.3 3.1 2.9 6.3 5.4 4.8 ND: NotDone RH: Relative Humidity

TABLE 2 CPE Bioassay Stability Data for the HA-Free Interferon beta 1bDrug Product Lot No.: 14159-49 25FEB2003 (Non-Clinical) (Test Run)TA2158 TA2451 Description: 1.2 ml; 0.25 mg/ml 1.2 ml, 1.0 mg/ml 1.2 ml,1.0 mg/ml 1.2 ml, 1.0 mg/ml Study No.: 1335 1438 1442 1444 Storage: 25°C./60% RH 25° C./60% RH 25° C./60% RH 25° C./60% RH Orientation:Inverted Inverted Inverted Inverted Months IU/mg × 10⁷ IU/mg × 10⁷ IU/mg× 10⁷ IU/mg × 10⁷ 0 2.8 2.7 2.7 2.6 1.5 3.2 3.7 3.5 3.6 3 4.3 4.4 4.44.2 4.5 4.1 4.8 4.6 4.6 6 4.6 5.3 5.5 5.4

As shown is Tables 3 and 4, below, cation exchange (CEX)-HPLCdemonstrates that the stability data for 25° C. samples of drugsubstance and drug product, respectively, also showed an increase indeamidation (D-IFN-β):

TABLE 3 CEX-HPLC Stability Data for the HA-Free Interferon beta 1b DrugSubstance Analytical Method: CEX HPLC Acceptance Criterion: Report %Deamidation Lot No.: Test Run TA2040 TA2085 Test Run TA2040 TA2085Description: 6 ml; 2 mg/ml 6 ml; 2 mg/ml 6 ml; 2 mg/ml 6 ml; 2 mg/ml 6ml; 2 mg/ml 6 ml; 2 mg/ml Study No.: 1402 1411 1414 1403 1412 1415Storage: 5° C. 5° C. 5° C. 25° C./60% 25° C./60% 25° C./60% RH RH RHOrientation: Upright Upright Upright Upright Upright Upright % % % % % %Months Deamidation Deamidation Deamidation Deamidation DeamidationDeamidation 0   6 6 6  6  6  6 1.5 ND ND ND 10 10  9 3   7 6 7 14 14 134.5 ND ND ND 20 20 18 6   5 6 6 22 22 20 ND: Not Done

TABLE 4 CEX-HPLC Stability Data for the HA-Free Interferon beta 1b DrugProduct Lot No.: 14159-49 25FEB2003 (Non-Clinical) (Test Run) TA2158TA2451 Description: 1.2 ml; 0.25 mg/ml 1.2 ml, 1.0 mg/ml 1.2 ml, 1.0mg/ml 1.2 ml, 1.0 mg/ml Study No.: 1335 1438 1442 1444 Storage: 25°C./60% RH 25° C./60% RH 25° C./60% RH 25° C./60% RH Orientation:Inverted Inverted Inverted Inverted Months % Deamidation % Deamidation %Deamidation % Deamidation 0 4  7  7  7 1.5 7 11 13 12 3 12 18 18 17 4.516 24 24 22 6 18   37 ¹   35 ¹   40 ¹ 10 END IP IP IP END

Tables 5 and 6, below, show that an increase in Peak D, which representsthe cyclic imide intermediate form of deamidation, was also observed inthe course of time by using a reversed-phase (RP)-HPLC technique in 25°C. samples of drug substance and drug product, respectively:

TABLE 5 RP-HPLC Stability Data for the HA-Free Interferon beta 1b DrugSubstance Analytical Method: RP HPLC Acceptance Criterion: Report % PeakAreas Lot No.: Test Run TA2040 TA2085 Description: 6 ml; 2 mg/ml 6 ml; 2mg/ml 6 ml; 2 mg/ml Study No.: 1403 1412 1415 Storage: 25° C./60% RH 25°C./60% RH 25° C./60% RH Orientation: Upright Upright Upright Main MainMain Months Pk A (D + B) Pk D Pk B Other Pk A (D + B) Pk D Pk B Other PkA (D + B) Pk D Pk B Other 0   2 96 12 85 2 2 96 11 85 2 2 96 11 86 2 1.52 95 17 78 3 2 96 17 79 2 2 96 16 80 3 3   1 93 20 73 6 1 92 19 73 7 192 19 73 7 4.5 2 91 24 67 7 2 90 24 66 8 1 91 23 68 8 6   2 93 26 67 5 193 25 68 5 1 94 25 68 5

TABLE 6 RP-HPLC Stability Data for the HA-Free Interferon beta 1b DrugProduct Analytical Method: RP HPLC Acceptance Criterion: Report % PeakAreas Lot No.: 25FEB03 TA2158 TA2451 (Test Run) Description: 1.2 ml, 1.0mg/ml 1.2 ml, 1.0 mg/ml 1.2 ml, 1.0 mg/ml Study No.: 1438 1442 1444Storage: 25° C./60% RH 25° C./60% RH 25° C./60% RH Orientation: InvertedInverted Inverted Main Main Main Months Pk A (D + B) Pk D Pk B Other PkA (D + B) Pk D Pk B Other Pk A (D + B) Pk D Pk B Other 0   1 93 12 81 61 93 11 82 6 1 93 12 81 6 1.5 1 92 16 76 6 1 92 17 74 7 1 91 17 75 8 3  2 90 22 68 8 2 91 21 69 8 2 91 21 70 7 4.5 2 94 23 71 5 1 94 22 72 5 194 22 72 4 6   1 94 24 70 5 1 94 24 71 5 1 95 24 17 4 END

FIGS. 4-11 show plots of the potency versus the amount of deamidatedIFN-β 1b (“% D-IFN” or “% Peak D”) in various HA-free IFN-β stabilitydrug substance and product lots in accordance with one aspect of theinvention. These data demonstrate that there is a correlation betweenthe potency increase and the level of deamidation of HA-free IFN-β.

2. Characterization of HA-Free IFN-β Stability Samples

The primary sequence of IFN-β 1b is shown in FIG. 3. Deamidation hasbeen found to occur at Asn25. The nature of that deamidation and theproperties of the deamidated products were explored in the assaysdescribed below:

2.1. Glu-C Peptide Map

A peptide map produces a fingerprint profile for proteins using anenzymatic digest followed by RP-HPLC. Peptide mapping is commonlyutilized in quality control as an ID test. It is also a powerful tool tomonitor minor primary structure modifications in a protein from eventssuch as clipping, mutation and degradation due to oxidation ordeamidation. HA-free IFN-β is known to contain cyclic imide, anintermediate form of deamidation. This cyclic imide is a key degradedvariant in HA-free IFN-β and is found in increased amounts in stabilitysamples. Most enzymes including Lys-C, which is used in the currentpeptide map for HA-free IFN-β, optimally digest proteins at neutral tohigh pH. At neutral to high pH, the cyclic imide is unstable anddeamidation is artificially induced. Therefore, maintaining the sampleat a low pH environment during reduction and digestion is necessary tomonitor the native level of cyclic imide and deamidation in the sample.

A new endoproteinase-C (Glu-C) peptide map has been developed coupledwith tris-(2-carboxyethyl) phosphine (TCEP) as reducing agent tocharacterize the fragment containing deamidated forms, including cyclicimide, in HA-free IFN-β. The Glu-C peptide map is known to have twooptimal pHs, pH 7.8 and pH 4.0, for its enzymatic activity. The TCEP isknown to be functional at pH below 8.0. The new Glu-C peptide map wasdeveloped using the sample preparation including both reduction anddigestion at low pH, 4.0, which is at the optimal pH range to preservethe native level of deamidated forms, e.g., cyclic imide, in the sample.Since the peptide map developed here employs a low pH samplepreparation, the accurate monitoring of the native level of deamidatedforms in HA-free IFN-β is successfully achieved.

Stability samples of HA-Free IFN-β 1b drug product lot TA2451 (3, 6, and9 months at 25° C.) were tested by Glu-C peptide map to identify thedeamidation site and its form (e.g., Asp, Iso-Asp, cyclic imide). Each0.5 ml protein sample at a concentration of 0.5 mg/ml in formulationbuffer of 2 mM aspartic acid, pH 4.0 was reduced using TCEP. Using a1:10 molar ratio of protein to TCEP, the sample was incubated at 37° C.for 3 hours. The reduced material was subsequently digested with 4 mg/mlGlu-C at a 5:1 mass ratio of protein to Glu-C and incubated at 37° C.for 4 hours. The peptide fragments were resolved by RP-HPLCchromatography (ThermoHypersil BioBasic C18, 150×4.6 mm, 5 μm), using anacetonitrile gradient in 0.1% trifluoroacetic acid as elution buffer, ata flow rate of 1.0 ml/min and a column temperature at 38° C.

FIG. 12A shows Glu-C peptide maps of HA-Free IFN-β stability samples inaccordance with one aspect of the invention. FIG. 12B shows an expandedview of the 22-26 minute RT portion of the map of FIG. 12A.

As shown in the figures, the peak of fragment E1 decreased in the courseof time, while the peaks representing iso-Asp, cyclic imide, and Aspincreased. Those peaks are deamidated forms of Asn at position 25 in thefragment E1. This peak characterization was performed by massspectrometry and Edman sequencing of E1 sub-fragments obtained by Lysylendopeptidase digestion of E1-related fragments. The Asn25 residue hasan amino acid sequence followed by glycine. This Asn-Gly sequence isknown to have a fast rate of deamidation.

Based on this Glu-C peptide map result, the major forms of deamidationin the HA-free IFN-β stability samples were identified to be iso-Asp andcyclic imide. There was also a slight increase in the level of the Aspform.

2.2. RP-HPLC

RP-HPLC separates compounds based on their hydrophobicity. HA-free IFN-β1b samples were tested by RP-HPLC method to characterize IFN-β 1bvariants (e.g., Asp, iso-Asp, cyclic imide). The test samples wereinjected onto a Zorbax 300SB-CN, 150×4.6 mm, 3.5 μm particle size,chromatography column and IFN-β 1b variants were separated using anacetonitrile gradient in 0.1% trifluoroacetic acid elution buffer. Thecontrol sample result is shown in FIG. 13.

The peak identification shown in the figure was performed by the onlineLC/mass spectrometric analysis (RP-HPLC/Q-TOF/ESI-MS). The HPLC flow wassplit approximately 1:20, and about 50 ul/min was directed to the ionsource of the mass spectrometer. The mass spectrometer was a MicromassQ-TOF2 instrument, with an electrospray ion source. The ion voltage wasset at 3200, with the cone voltage set at 50. Data was collected withthe time-of-flight (TOF) analyzer between m/z 300 and 2500. The onlineLC/mass data reveals that there is more than one protein variantco-eluting under some of the RP-HPLC peaks.

FIG. 14 shows the RP-HPLC profile of the stability sample of the HA-freeIFN-β 1b drug product lot (about 10 months at 25° C.). As shown in thefigure, the profile is different from that of control sample (FIG. 13)and cyclic imide (Peak D) and deamidation (Asp and/or iso-Asp) (MidPeak) significantly increased in HA-free IFN-β 1b. Multiple minor otherdeamidated and cyclic imide variants are observed. These are deamidatedand cyclic imide variants with additional structural and chemicalmodifications that are separated due to their differing hydrophobicproperties.

2.3. CPE Bioassay

IFN-β induces an antiviral state in mammalian cells in which some virustypes are inhibited from replicating and causing cellular cytopathiceffects (CPE). A549 human lung carcinoma cells and murineencephalomyocarditis (EMC) virus were used to evaluate biologicalactivity of the RP-HPLC fractions obtained from the stability sample ofthe HA-free IFN-β 1b drug product lot (about 10 months at 25° C.).

Cells were grown in 96-well plates and treated with serial dilutions ofIFN-β 1b overnight before addition of virus. Cultures were thenincubated for a suitable period of time to allow virus replication.Cells treated with sufficient IFN-β 1b were protected from the viruschallenge and remain viable. Unprotected cells underwent cytopathicchanges and died. The interferon dose dependent CPE was quantitatedusing staining techniques and a dose response curve prepared from a plotof cell viability (Optical Density measurement) vs. IFN-β concentration.IFN-β activity was calculated as the concentration required for halfmaximal cell protection (ED₅₀ concentration).

As shown in FIG. 15, the CPE activity in the deamidated fraction (MidPeak, Fraction 16) is significantly higher than that in the parent IFN-β1b fraction (Peak B, Fraction 18). The cyclic imide fraction (Peak D,Fraction 15) also shows higher CPE activity than the parent IFN-β 1bfraction.

2.4. Antiproliferative Assay

IFN-β shows antiproliferative activity against a number of cell linesestablished from human tumors. Two cell lines, Hs294T-a human melanomacell line and Daudi-a human B lymphoblast line derived from Burkittlymphoma, were used to evaluate biological activity of the RP-HPLCfractions obtained from the stability sample of the HA-free IFN-β 1bdrug product lot (about 10 months at 25° C.).

Serial dilutions of the IFN-β test samples were performed in 96-wellplates. Responder cells were prepared in tissue culture medium and addedto the assay plates. After 3 days incubation, staining the cells withAlamar Blue or counting with a Coulter counter was used to measure thegrowth response. Cell growth was inhibited in response to IFN-β in adose dependent manner. A dose response curve was prepared from a plot ofcell number (Optical Density measurement) vs. IFN-β concentration. IFN-βactivity was calculated as the concentration required for half maximalcell growth (ED₅₀ concentration).

As shown in FIGS. 16A (Hs294T) and 16B (Daudi), the antiproliferativeactivity in the deamidation fraction (Mid Peak, Fraction 16) and cyclicimide fraction (Peak D, Fraction 15) is significantly higher than thatin the parent IFN-β fraction (Peak B, Fraction 18).

3. Conclusions

Based on the stability data and results obtained from studies describedabove, deamidation (Asp, iso-Asp and cyclic imide replacing Asn atposition 25) enhances a biological activity of IFN-β. Therefore, adeamidated form of an IFN-β, e.g., IFN-β 1b can be intentionallyprepared in order to enhance the compound's biological activity. Thedeamidated form can be prepared by incubating IFN-β at moderate to hightemperature, or at low, moderate or high pH environment. The deamidatedproducts of this invention will reduce the required clinical dose andincrease the stability of liquid IFN-β formulations, including HA-freeand HA-containing IFN-β formulations, at room temperature. By loweringthe clinical dose, the proportion of patients experiencing an adverseimmune reaction (e.g., neutralizing antibodies), is reduced.

Example 2 Manufacturability Data

Experiments were conducted to demonstrate various deamidationtechniques, as described above. The data obtained from the CPE bioassayevidences that deamidated IFN-β is readily prepared by processessuitable for large scale pharmaceutical production. In particular,significant bioactivity increases were observed in various samplesmanufactured under Good Manufacturing Practices (GMP) incubated atmoderate (e.g., room temperature) to high (e.g., 37-40° C.) temperaturefor 14-40 days, and bioactivity increases of almost 2 fold were obtainedby incubating a GMP sample at room temperature and pH 8.8 for about 1hour, and at 2-8° C. at pH 8.4 for about 14 days.

CPE Bioassay Results

Sample Name IU/mg Test 1 TR090602 Bulk 2.79E+07 TR090602 Stab RT¹ 20days 3.87E+07 TR090602 Stab 37 C. 20 days 4.11E+07 Stressed HA FreeControl 2.95E+07 Stressed HA Free Base pH² 4.52E+07 Stressed HA Free 40C. 30 Days 5.06E+07 IFN CPE Assay Control 2.94E+07 Test 2 TR090602 Bulk3.62E+07 TR090602 Stab RT 40 days 6.52E+07 TR090602 Stab 37 C. 40 days1.56E+08 GMP#1 Bulk 2.67E+07 GMP#1 Bulk Stab 37 C. 40 days 3.51E+07GMP#1 Bulk pH 8.8³ 4.79E+07 Stressed HA free Control 2.26E+07 StressedHA free 40 C. 14 days 3.44E+07 Stressed HA free 40 C. 30 days 4.05E+07IFN CPE Assay Control 2.93E+07 ¹Room temperature ²Sample was incubatedin pH 8.4 at 2-8 C. for 14 days. ³Sample was incubated in pH 8.8 at RTfor 1 hr.

CONCLUSION

Although the foregoing invention has been described in some detail forpurposes of clarity of understanding, it will be apparent that certainchanges and modifications may be practiced within the scope of theappended claims. It should be noted that there are many alternative waysof implementing both the processes and compositions of the presentinvention. Accordingly, the present embodiments are to be considered asillustrative and not restrictive, and the invention is not to be limitedto the details given herein, but may be modified within the scope andequivalents of the appended claims.

All documents cited herein are hereby incorporated by reference hereinin their entirety and for all purposes.

1. A purified and isolated synthetic human interferon-β protein analog,wherein, the asparagine at position 25, numbered in accordance withnative interferon-β, is deamidated, and, said protein analog exhibits abiological activity of native human interferon-β.
 2. The syntheticprotein analog of claim 1, wherein the cysteine at position 17, numberedin accordance with native interferon-β, is deleted or replaced by aneutral amino acid.
 3. The synthetic protein analog of claim 2, whereinsaid cysteine residue has been replaced by a serine residue.
 4. Thesynthetic protein analog of claim 3, wherein said asparagine residue hasbeen replaced by a residue selected from the group consisting ofaspartate, iso-aspartate and cyclic imide.
 5. The synthetic proteinanalog of claim 4, wherein the protein analog is unglycosylated.
 6. Thesynthetic protein analog of claim 5, wherein the protein analog has anN-terminal methionine deletion.
 7. The synthetic protein analog of claim4, wherein the protein analog has a biological activity greater thanIFN-β_(ser17).
 8. A therapeutic composition having IFN-β activitycomprising a therapeutically effective amount of the synthetic proteinanalog of claims 4 admixed with a pharmaceutically acceptable carriermedium.
 9. The composition of claim 8, wherein at least 50% of thesynthetic protein analog is deamidated at position 25, numbered inaccordance with native interferon-β.
 10. The composition of claim 8,wherein substantially all of the synthetic protein analog is deamidatedat position 25, numbered in accordance with native interferon-β.
 11. Thecomposition of claim 8, wherein the composition is HA-free.
 12. A methodof making deamidated INF-β analog, comprising: incubating an IFN-βprotein under conditions suitable at moderate to high temperature ofabout 25-60° C.; purifying and isolating the deamidated protein analog.13. The method of claim 12, comprising: incubation at a temperature ofabout 40° C. for about 14 days.
 14. The method of claim 13, furthercomprising wherein the incubation is conducted at a pH of at least 4.15. The method of claim 14, comprising: incubation at a pH of 7-14. 16.The method of claim 15, wherein the incubation is at pH 8-9 for about 14days.
 17. A method of treating a patient comprising administering tosaid patient an effective amount of the composition of claim
 8. 18. Themethod of claim 17, wherein the treatment is for regulating cell growthin the patient and the effective amount is a cell growth regulatingamount of the composition.
 19. The method of claim 17, wherein thetreatment is for a viral disease of the patient and the effective amountis a viral disease inhibiting amount of the composition.
 20. The methodof claim 17, wherein the treatment is for stimulating natural killercell activity in the patient and the effective amount is a naturalkiller cell stimulating amount of the composition.
 21. The method ofclaim 17, wherein the treatment is for multiple sclerosis in the patientand the effective amount is a therapeutically effective amount of thecomposition.
 22. The method of claim 21, wherein the treatment comprisesreducing the frequency of multiple sclerosis flare-ups.
 23. The methodof claim 22, wherein said multiple sclerosis is relapsing remittingtype.
 24. A peptide mapping method, comprising: incubating a proteinsample in a buffered solution below pH 8 containing a reducing agent;digesting the incubated sample with endoproteinase-C; and resolving thepeptide fragments of the digest by liquid chromatography.
 25. The methodof claim 24, wherein the reducing agent is tris-(2-carboxyethyl)phosphine (TCEP).
 26. The method of claim 25, wherein the incubation isconducted at a pH of about
 4. 27. The method of claim 26, wherein theliquid chromatography is RP-HPLC.
 28. The method of claim 27, whereinthe protein sample is a deamidated IFN-β.